Intranasal immunisation with recombinant Toxoplasma gondii uridine phosphorylase confers resistance against acute toxoplasmosis in mice

Toxoplasmosis is caused by Toxoplasma gondii, which infects all warm-blooded animals, including humans. Currently, control measures for T. gondii infection are insufficient due to the lack of effective medications or vaccines. In this paper, recombinant T. gondii uridine phosphorylase (rTgUPase) was expressed in Escherichia coli and purified via Ni2+-NTA agarose. rTgUPase was inoculated intranasally into BALB/c mice, and the induced immune responses were evaluated by mucosal and humoral antibody and cytokine assays and lymphoproliferative measurements. Moreover, the protective effect against the T. gondii RH strain infection was assessed by calculating the burdens of tachyzoites in the liver and brain and by recording the survival rate and time. Our results revealed that mice immunised with 30 μg rTgUPase produced significantly higher levels of secretory IgA (sIgA) in nasal, intestinal, vaginal and vesical washes and synthesised higher levels of total IgG, IgG1 and, in particular, IgG2a in their blood sera. rTgUPase immunisation increased the production of IFN-gamma, interleukin IL-2 and IL-4, but not IL-10 from isolated mouse spleen cells and enhanced splenocyte proliferation in vitro. rTgUPase-inoculated mice were effectively protected against infection with the T. gondii RH strain, showing considerable reduction of tachyzoite burdens in liver and brain tissues after 30 days of infection, and a 44.29% increase in survival rate during an acute challenge. The above findings show that intranasal inoculation with rTgUPase provoked mucosal, humoral and cellular immune responses and indicate that rTgUPase might serve as a promising vaccine candidate for protecting against toxoplasmosis.


Introduction
Toxoplasmosis is a disease caused by the protozoan parasite Toxoplasma gondii, which infects approximately one-third of humans [23].Usually, toxoplasmosis in immunocompetent people is asymptomatic, whereas it seriously affects immunocompromised individuals, such as AIDS patients, cancer patients, and organ transplant recipients, causing serious medical issues [30].In particular, this disease may lead to foetal mortality in cases of vertical passage [1].Unfortunately, current therapeutic options for toxoplasmosis are limited.Antifolate drugs such as pyrimethamine are effective against the tachyzoite stage of T. gondii but do not affect the bradyzoite stage that causes chronic infection in the host.Lifelong maintenance with a combination of pyrimethamine-sulfadiazine for toxoplasmic encephalitis often leads to side effects, including severe allergic reactions and haematoxicity [10].Vaccine inoculation is a promising method in therapeutic applications of toxoplasmosis since vaccination is the most effective and potent strategy for controlling infectious diseases and has saved millions of lives [4].Consequently, finding and validating vaccine candidates against toxoplasmosis is especially urgent.
Over the past few years, protein vaccines have received increasing attention since they can provoke strong humoral responses by eliciting antigen-specific antibodies [36].Furthermore, the non-invasive and acceptable route of intranasal vaccination is becoming more attractive because it requires lower doses of antigens, while it triggers mucosal immune responses at local and distant mucosa sites, as well as systemic and cellular immune response [27].Therefore, several recombinant T. gondii proteins prepared by other groups such as rhoptry protein 2 (ROP2) [13], ROP18 [31], malate dehydrogenase (MDH) [19], actin depolymerizing factor (ADF) [18], as well as ours such as actin [44], phosphoglycerate mutase 2 (PGAM 2) [41], ROP17 [42], receptor for activated C kinase 1 (RACK1) [39] and protein disulfide isomerase (PDI) [38] have been tested to assess their immunoprotective effects produced by intranasal immunisation.Although the abovementioned recombinant proteins were capable of triggering both cellular and humoral responses, they all conferred partial protective efficacy against toxoplasmosis.
Toxoplasma gondii is a member of the phylum Apicomplexa, which replicates rapidly and requires large amounts of purines for the synthesis of their nucleic acids and other vital components.Nevertheless, it is a purine auxotroph that relies on purine salvage from the host [8].Pyrimidine salvage in T. gondii probably occurs through the following steps: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively, uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase (UPase), and uracil is metabolised to uridine 5 0 -monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5 0 -monophosphate is the end product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii [14].UPase participates in the synthesis of purines and plays an important role in the proliferation of T. gondii.
Our previous study showed that T. gondii UPase is one of the novel candidate antigens identified among soluble tachyzoite antigens using rabbit anti-T.gondii serum by twodimensional gel electrophoresis and proteomics analyses [20].The recombinant T. gondii uridine phosphorylase (rTgUPase) protein was produced in Escherichia coli and showed specific antigenicity [45].In the present study, rTgUPase was used to intranasally immunise BALB/c mice, and the immune protection against T. gondii infection was investigated.The results demonstrated that mice immunised with rTgUPase could protect against T. gondii infection by eliciting humoral and mucosal as well as cellular immune responses.Additionally, our data showed that rTgUPase may be a novel vaccine candidate against toxoplasmosis.

Mice, ethics statement and parasites
Female BALB/c mice (6-week-old) were purchased from the Laboratory Animal Center, Shanxi Medical University (Shanxi, China).All mice were maintained under specificpathogen-free (SPF) conditions and provided with rodent feed and water ad libitum.Prior to experiments, the mice were acclimatised for one week.The animal protocols were approved by the Ethics Committee on Animal Research of the Shanxi Medical University (Protocol #: SYDL2021016).
Tachyzoites of the virulent RH strain of T. gondii used as a challenge for immunised mice were provided by Peking University Health Science Center (Beijing, China) in this study.Tachyzoites of the highly virulent T. gondii RH strain (Type I) were maintained using Vero cells in MEM with 5% FBS (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA).The T. gondii tachyzoites were cultivated every 4 days in Vero cells and collected according to published protocols [25,26].
Expression and purification of rTgUPase rTgUPase was expressed in E. coli strain BL21 (DE3) and purified via affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) agarose (QIAGEN, Hilden, Germany) as described previously [45].Briefly, total RNA was extracted from tachyzoites of the RH strain of T. gondii.A pair of specific primers (sense, 5 0 -CCGGAATTCATGTCGGAACTCAAAG-GAA-3 0 ; antisense, 5 0 -CCGGCTCGAGTT ACGCCGCAGG CTTGATG-3 0 ) were designed according to the open reading frame of the TgUPase gene (GenBank: DQ385446.1),and the RT-PCR product was cloned into the prokaryotic expression pET-30a(+) vector.The recombinant pET-30a(+)-TgUPase Herein, cultured pET-30a(+)-TgUPase-BL21 (DE3) with or without IPTG induction served as induced or uninduced group, respectively.The bacteria from 1 mL induced or uninduced group were collected by centrifugation and boiled in 100 lL 1 Â SDS loading buffer for 5 min, then 10 lL was loaded on SDS-PAGE.The bacteria from 3 mL induced group were harvested via centrifugation, and the obtained pellets were resuspended in cold PBS and homogenised via sonication on ice.The lysate was then centrifuged to separate the supernatant and cell pellet.The cell pellet was boiled in 100 lL 1 Â SDS loading buffer and then 10 lL was loaded on SDS-PAGE.The supernatant was quantified using BCA method and 10 lg protein was loaded on SDS-PAGE.

rTgUPase immunisation and sample collection
Forty female BALB/c mice were randomly divided into five groups (8 per group) and intranasally immunised with 20 lL of PBS containing 10 lg, 20 lg, 30 lg or 40 lg rTgUPase on Days 0, 14 and 21.The control group was treated with 20 lL of PBS instead.Two weeks after the third inoculation, the mice were anaesthetised with sodium pentobarbital (1.5%, 0.1 mL/20 g weight, intraperitoneal injection), and blood samples from mice in each group were collected by retro-orbital plexus puncture.The sera were separated and stored at À70 °C until analysed for the presence of specific antibodies.
The spleens were collected under aseptic conditions to perform lymphocyte proliferation assays, and the culture supernatants were used for cytokine assays.Prior to sample collection, the mice were deprived of food for 8 h to deplete the intestinal contents.Nasal, intestinal, vaginal and vesical washes were collected according to a previously described method [44,47].All the samples were stored at À70 °C for secretory IgA (sIgA) assays.

Spleen lymphocyte proliferation assay
According to our previously described method [44], spleens were surgically removed from the mice, and single-cell preparations were pelleted on Day 15 after the last immunisation.
In brief, 5 Â 10 5 cells per well were cultured in triplicate in 96-well plates containing RPMI-1640 medium with penicillinstreptomycin (1 mM) and 10% FBS.The culture was stimulated with either 10 lg/mL rTgUPase, 5 lg/mL concanavalin A (Con A) as a positive control or medium alone for proliferation.The plates were incubated in 5% CO 2 at 37 °C for 4 days.Next, 10 lL of CCK-8 reagent (Dojindo Laboratories, Japan) was added to each well, and the plate was incubated for 3 h.The optical density was then determined at 450 nm using an ELISA reader.The spleen cell proliferative responses were quantitated using a stimulation index (SI), which was calculated as the ratio of the average OD 450 of the stimulated cells to the average OD 450 of the unstimulated cells.All assays were performed in triplicate.

Cytokine assays
Cytokines were measured as previously described [40].Spleen cells were obtained as described above and cultured in triplicate in flat-bottom 24-well microtiter plates.Supernatants from the cultured splenocytes (1.5 Â 10 6 ) were collected after 24, 72 or 96 h of stimulation with rTgUPase (10 lg/mL) and assayed for interleukin-2 (IL-2) and IL-4 at 24 h, for IL-10 at 72 h, and for interferon-gamma (IFN-c) at 96 h.IL-2, IL-4, IL-10 and IFN-c concentrations were determined using a commercial ELISA Kit (PeproTech, USA), according to the manufacturer's instructions.Cytokine concentrations were determined by reference to standard curves constructed with known amounts of mouse recombinant IL-2, IL-4, IL-10 and IFN-c.The sensitivity limits of detection of IL-2, IL-4, IL-10 and IFN-c were 16, 16, 47 and 23 pg/mL, respectively.

Specific IgG and sIgA detection
Enzyme-linked immunosorbent assays (ELISAs) were performed for the detection of rTgUPase-specific IgG, IgG1 and IgG2a antibodies in serum samples and sIgA in nasal, intestinal, vaginal and vesical washes collected two weeks after the last immunisation according to our previously described method [41].Briefly, 96-well flat-bottom microtiter plates were coated with 7.5 lg/mL rTgUPase (100 lL/well) in PBS overnight at 4 °C.The plates were washed with PBS containing 0.05% Tween 20 (PBST), blocked for 1 h at 37 °C in PBS containing 5% FBS, and then washed with PBS.Thereafter, the serum samples (1:200 for IgG, 1:50 for IgG1 and IgG2a) and mucosal washes were incubated in different wells (100 lL/well) for 1 h at 37 °C.After washing, the wells were incubated with 100 lL of goat anti-mouse HRP-IgG, HRP-IgG1, HRP-IgG2a or HRP-IgA (Proteintech, China; diluted 1:2000 in PBS) for 1 h at 37 °C.The plates were washed extensively and incubated with 100 lL of substrate solution for 30 min at 37 °C.The optical density was measured at 492 nm (OD 492 ) with a microplate reader (Bio-Tek) followed by 50 lL of 2 N H 2 SO 4 to stop the enzyme reaction.All the samples were run in triplicate.

Challenge infection
Two groups of 6-week-old female BALB/c mice (20 mice per group) were vaccinated intranasally with 30 lg of rTgUPase suspended in 20 lL of sterile PBS or 20 lL of PBS and boosted with the same dose three times on Days 0, 14 and 21.The rTgUPase immunisation dose and the immune programme were based on the results of the abovementioned experiment.On Day 15 after the last immunisation, 8 mice in each group were challenged orally with 1 Â 10 4 tachyzoites of the RH strain for the tachyzoite-load assay, while 12 mice were challenged with 4 Â 10 4 tachyzoites for lethal infection.
On the 30th day after being challenged, the infected mice were anaesthetised with sodium pentobarbital, and the numbers of tachyzoites in the livers and brains were measured using realtime PCR assays as previously described [38,39,41,44].For survival analysis, the acute infected mice were monitored thrice daily until 30 days after the tachyzoite challenge.When painful symptoms were observed, a mouse was moved to an isolated cage for further husbandry; if obvious suffering, such as struggling or whining, was observed, the mouse was sacrificed through carbon dioxide (CO 2 ) inhalation.

Statistical analysis
All statistical analyses were performed using SPSS software for Windows version 19.0.The differences in each variable, including antibody responses, lymphoproliferation assays and cytokine production, among all the groups were compared by one-way ANOVA.The tachyzoite burdens and survival times for vaccinated and control mice were compared using the Kaplan-Meier method.Significant differences in comparisons between groups were defined at p < 0.05.

Results rTgUPase was expressed and purified successfully
After induction with 0.1 mM IPTG at 37 °C, the rTgUPase proteins were successfully expressed in E. coli, and the molecular weight was approximately 38.0 kDa (Fig. 1A).The isolated protein was water soluble and showed greater than 95% purity based on SDS-PAGE analysis (Fig. 1B).Western blot analysis indicated that the rTgUPase band was able to react with anti-Toxoplasma human serum (Fig. 1C).

Systemic immune response induced by rTgUPase vaccination
To assess the systemic immune response in the immunised mice, we evaluated the levels of rTgUPase-specific IgG, IgG1 and IgG2a antibodies in the sera and cytokines from the spleen cell supernatants by ELISAs.The results showed that 20, 30 and 40 lg of rTgUPase could elicit the maximum IgG antibody responses compared to those of the PBS and 10 lg groups (p < 0.01), but no significant differences were observed in the IgG responses among the 20, 30 and 40 lg groups and between the PBS and 10 lg groups (p > 0.05) (Fig. 2A).A mixed IgG1/IgG2a response was detected in the sera of the mice immunised with rTgUPase (Fig. 2B).Moreover, the mice immunised with 20, 30 and 40 lg rTgUPase elicited higher levels of IgG1 and IgG2a than the controls (p < 0.01).The 30 lg group had the highest value but was not significantly different from the 20 and 40 lg groups (p > 0.05).The above findings suggested that rTgUPase intranasal immunisation provoked a mixed Th1/Th2 immune response.

Cellular immune response elicited by rTgUPase inoculation
Spleen cells from 5 groups of mice were prepared 2 weeks after the last immunisation to assess the proliferative responses to rTgUPase.As shown in Table 1, the splenocyte stimulation indices (SI) from the immunised groups were higher than that of the PBS group (p < 0.05; p < 0.01), and the 30 lg group had the strongest activity vs. all other groups.In addition, splenocytes from each experimental and control group proliferated well in response to ConA (data not shown).
The cell-mediated immunity produced in the immunised mice was evaluated by measuring the amount of cytokines (IFN-c, IL-2, IL-4 and IL-10) in the supernatants of stimulated splenocyte cultures from mice of all groups.As shown in Table 1, significantly higher levels of IFN-c, IL-2 and IL-4 were detected in the mice from all immunised groups compared with the controls (p < 0.05; p < 0.01).The mice immunised with 30 lg had the highest levels of IFN-c, IL-2 and IL-4 compared to those of the other dose groups.However, the production of IL-10 did not significantly differ among all groups (p > 0.05).

Mucosal immune responses induced by rTgUPase vaccination
To investigate whether the mice immunised with rTgUPase induced mucosal immune responses, we tested the levels of rTgUPase-specific sIgA in the mucosal washes by ELISAs two weeks after the last immunisation (Fig. 3).The titres of rTgUPase-specific sIgA antibody in the mucosal washes were elevated following nasal immunisation.The sIgA antibody titres in the intestinal washes of the 10, 20, 30 or 40 lg rTgU-Pase-treated groups were significantly higher than those of the PBS control (p < 0.01), with the highest titre of sIgA antibody detected in the 30 lg rTgUPase group (Fig. 3B).sIgA levels from the nasal, vaginal and vesical washes were higher in the mice that were nasally immunised with 20, 30 or 40 lg rTgU-Pase compared with those from the PBS control and 10 lg rTgUPase groups, and 30 lg rTgUPase also provoked the highest sIgA levels in nasal washes (p < 0.01), while 40 lg rTgU-Pase induced the highest sIgA levels in vaginal and vesical washes (p < 0.01) (Figs.3A, 3C and 3D).No significant differences were found between the mucosal washes of the 30 and 40 lg rTgUPase-treated groups.In conclusion, strong mucosal immune responses were elicited by nasal immunisation with rTgUPase at nasal, intestinal, vaginal and vesical mucosal sites.

Protection against T. gondii infection
To estimate the protective efficacy of rTgUPase intranasal immunisation against T. gondii infection, we generated a mouse model of tachyzoite infection via the oral route according to published procedures [41].Thirty days after the challenge, the tachyzoite loads in the brain tissues were 18.18 (± 1.03) Â 10 6 /g in the control group and 9.47 (± 0.16) Â 10 6 /g in the rTgUPase-vaccinated group.The tachyzoite loads in the liver tissues were 62.52 (± 8.96) Â 10 6 /g in the control group and 30.15 (± 6.46) Â 10 6 /g in the rTgUPase-vaccinated group.These data indicated that immunisation with 30 lg of rTgUPase markedly reduced the tachyzoite loads compared to those in the control mice, showing approximately 51.78% (p < 0.01) and 49.61% (p < 0.05) fewer tachyzoites in the liver and brain, respectively (Fig. 4A).
Additionally, the survival rates of the mice were recorded daily following oral challenge (4 Â 10 4 tachyzoites of the RH strain) until 30 days post-challenge.A significant increase in the survival rate was observed in the 30 lg rTgUPase-immunised group compared to the control group (P < 0.01) (Fig. 4B).The mice immunised with 30 lg of rTgUPase had a significantly increased survival rate (44.29%) on the 30th day after challenge, and all mice in the PBS group died within 12 days post-challenge.These results demonstrated the protective effect of rTgUPase against T. gondii RH strain challenge.

Discussion
Toxoplasma gondii is an obligate intracellular protozoan that infects almost all warm-blooded animals, including humans.There are three asexual stages, including sporozoite,  bradyzoite and tachyzoite, that can invade the cells in the T. gondii life cycle [22].During these asexual stages, sporozoites are produced by sexual reproduction and excreted in the oocysts through felid faeces; bradyzoites are a slow multiplication form coming from the tissue cysts when chronic infection occurs; and tachyzoites, a proliferative form that causes a decrease in cholesterol content in the liver and brain and a decrease in host immune functions, leading to acute infection [37].Importantly, tachyzoites can be maintained and produced in vitro (cellular cultivation systems) and in vivo (animal models), which makes them the most experimentally manipulable form that has been extensively modelled in many studies, including antigen production, immunological studies, drug trials in vitro and others [3].Given these aspects of the tachyzoite, in the present study, T. gondii-infected mouse models were employed using tachyzoites.Of course, another consideration is that the antigen rTgUPase, which converts uridine or deoxyuridine to uracil and participates in the pyrimidine salvage pathways of T. gondii, originates from tachyzoites [11,20].TgUPase has been identified from soluble tachyzoite antigens and can react with a rabbit anti-T.gondii serum [20].Moreover, rTgUPase could react with human anti-T.gondii serum [45], indicating that this protein could be used as a novel vaccine candidate.Here, rTgUPase nasal inoculation in BALB/c mice enhanced the output of sIgA in mucosal tracks and the production of total IgG and IgG isotypes (IgG1 and IgG2a) in sera.Additionally, rTgUPase stimulated lymphocyte proliferation and the release of the cytokines IFN-c, IL-2, and IL-4 but not IL-10 in isolated spleen cells from inoculated mice.Decreased loads of tachyzoites in host tissues, an enhanced host survival rate (44.29%) and prolonged survival time were observed in the rTgUPase-vaccinated mice challenged with T. gondii infection.These data demonstrate that rTgUPase is a promising immunogen for developing a mucosal vaccine against T. gondii infection.
The natural portal of infection of T. gondii is the oral route by which either cyst-contaminated meats or oocyst-polluted fruits and vegetables are ingested [43].In previous papers, tachyzoites have been proven to infect cats and mice via the oral route [9,29,41].After ingestion, T. gondii targets the small intestine for infection.Given the mucosal exposure to Toxoplasma, mucosal immunity is believed to protect against Toxoplasma infection [46].IgA is the most abundant antibody isotype in mucosal immunity to many pathogens, including Toxoplasma, which invades the host organism by crossing mucous membranes [12].sIgA, which is the major effective form of mucosal IgA, annihilates pathogens with immune exclusion via nonspecific immunity [6].Additionally, sIgA plays an indispensable role in specific immunity provoked by pathogens or mucosal vaccines [16].At the site of entry, sIgA, which is synthesised from the epithelial cells of the intestine, can partially eliminate T. gondii via nonspecific immune exclusion and/or a specific neutralising role [16].Our present results revealed that sIgA antibodies in intestinal washes as well as in nasal, vaginal and vesical washes were significantly enhanced in 30 lg rTgUPase-vaccinated mice.These data indicate that local mucosal inoculation with recombinant T. gondii protein can elicit in situ and distal immune responses.Therefore, mucosal inoculation via the intranasal route has extensive potential for sparking a protective immune response in all mucosal compartments.
Apart from sIgA, IgG, another element in humoral immunity, also participates in resistance to T. gondii [35].The isotypes IgG, IgG1 and IgG2a can play an important role in resistance to T. gondii through complement fixation, opsonisation, or antibody (Ab)-dependent cell cytotoxicity [7].Here, immunisation of mice with 30 or 40 lg of rTgUPase led to the development of higher levels of total rTgUPase-specific IgG antibodies compared with those of the PBS and 10 and 20 lg groups.Furthermore, a mixed humoral response of both IgG1 and IgG2a was unveiled in the 30 or 40 lg rTgUPase-vaccinated mice, indicating that rTgUPase immunisation predominantly activated a mixed Th1/Th2 immune response.
Cell-mediated immunity plays a major role in anti-T.gondii immunity.Among the cytokines produced by activated immune cells such as natural killer (NK) cells and CD4 + and CD8 + T lymphocytes, IFN-c plays a pivotal role in the host defence against T. gondii infection [33].IFN-c controls tachyzoite replication during both acute and chronic phases of infection and prevents reactivation of T. gondii from dormant cysts at a later phase [2].IFN-c also promotes robust production of indoleamine 2,3-dioxygenase (IDO), which suppresses T. gondii growth [24].In addition, IFN-c-stimuslated cells express copious IFN-stimulated proteins, including GTPase family members, such as immunity-related GTPases (IRGs), which accumulate on the T. gondii PV membrane (PVM), destroy this structure, and then kill the parasites [32].IL-2 can drive NK and CD8 + cell expansion, which is responsible for controlling tachyzoite proliferation via the synthesis of IFN-c [34].IFN-c and IL-2 can stimulate the induction of antigen-specific sIgA responses that control the invasion of T. gondii at intestinal mucosal sites [39].IL-4 plays a major role in controlling the development of cell-mediated immunity (CMI) and is involved in protection against the development of toxoplasmic encephalitis by preventing the formation of T. gondii cysts and the proliferation of tachyzoites in the brain [5].IL-10 is a major antagonist involved in modulating IFN-c, avoiding an extreme immune response that causes extensive inflammation and host tissue damage [28].In our study, the production of IFN-c, IL-2 and IL-4 rather than IL-10 in the supernatant of cultured spleen cells was significantly increased in the rTgUPasevaccinated mice, suggesting that Th1-and Th2-type cellularmediated immune responses were generated.Furthermore, the elevated number of spleen lymphocytes was consistent with the augmentation of cytokines in different groups stimulated with rTgUPase.These cytokines are valid evaluation indicators for the actual response in an in vivo assay to assess the immune responses of novel vaccine candidates against toxoplasmosis [5].The above findings suggested that rTgUPase intranasal inoculation could elicit cell-mediated immune responses.
Furthermore, we prepared T. gondii RH strain infection mouse models via peroral infection of tachyzoites to assess the protective effect of the rTgUPase protein against T. gondii infection.Obvious reductions in tachyzoites were observed in the liver and brain tissues of the rTgUPase-vaccinated mice compared with those in the control mice.Additionally, the immunised mice exhibited significant protection against lethal tachyzoite infection, showing an approximately 44.29% improved survival rate and prolonged lifespan.In conclusion, TgUPase immunisation is effective in decreasing the loads of tachyzoite infection in host tissues and partially protects the host against T. gondii infection.Apart from type I strains, type II strains have low virulence, and type III strains are avirulent [11].For further evaluation of the protective effect of rTgUPase, a type II or type III strain of T. gondii should be used to challenge mice in the next study.
To date, a T. gondii vaccine for clinical application is not yet available, although multiple vaccine candidates have been suggested.The reason is the existence of both multiple antigenically distinct strain types and multifarious antigenically diverse developmental stages.Moreover, Toxoplasma can effectively escape the immune system by developing a chronic encysted stage that is not easily eliminated by immune responses.An effective vaccine can prevent the tachyzoites from gaining access to other host tissues and to the placenta.Additionally, this vaccine can cause an immune response able to stop and kill the parasite when it penetrates the intestinal barrier.Of course, an effective vaccine must induce an immune response able to inhibit the formation of tissue cysts during the chronic phase of toxoplasmosis.For these demands, multicomponent antigens from oocysts, bradyzoites, tachyzoites and cysts should be considered.In addition, appropriate adjuvants such as CpGcontaining oligodeoxynucleotide (CpG ODN) [15] and nanocurcumin [21] should be taken into account.
Taken together, our findings indicate that intranasal inoculation with rTgUPase provokes both mucosal and systemic as well as cellular-mediated immune responses, which confer protection against T. gondii RH strain infection in BALB/c mice.Although the protective effect is partial, findings from the present study indicate that rTgUPase is a promising vaccine candidate against Toxoplasma.

Figure 1 .
Figure 1.SDS-PAGE and Western blot analyses of the rTgUPase protein.(A) The full-length ORF of TgUPase was expressed in E. coli, separated on 12% SDS-PAGE gels and stained with Coomassie blue.The molecular weight of rTgUPase was approximately 38.0 kDa.(B) Purified rTgUPase protein (2 lg/lane) was detected via 12% SDS-PAGE and stained with Coomassie blue, and the purity of rTgUPase was greater than 95%.(C) Western blot analysis of rTgUPase using a human anti-T.gondii serum.Left lane: the supernatant (10 lg), Middle lane: cell pellet, right lane: induced whole bacterial protein.

Figure 2 .
Figure 2. Nasal immunisation induces rTgUPase-specific IgG responses in sera.Titres of both specific total IgG and IgG isotype antibodies in the sera of BALB/c mice were determined by ELISAs with rTgUPase as the bound target two weeks after the last immunisation.(A) Specific total IgG and (B) IgG1 and IgG2a titres in the sera of the mice vaccinated with rTgUPase.The results are expressed as the means of the OD 492 ± SD (n = 8) and are representative of three experiments.** p < 0.01 (vaccinated vs. PBS group).

Figure 3 .
Figure 3. Nasal immunisation induces rTgUPase-specific sIgA responses in mucosal washes.The sIgA antibody titres in mucosal washes from the mice were tested by ELISAs two weeks after the last immunisation.High-level sIgA in (A) nasal washes, (B) intestinal washes, (C) vaginal washes and (D) vesical washes was induced in the mice nasally immunised with rTgUPase compared to those vaccinated with PBS.The data are expressed as the means of the OD 492 ± SD (n = 8) and are representative of three experiments.*p < 0.05, ** p < 0.01 (vaccinated vs. PBS group).

Figure 4 .
Figure 4. Assay for protection against oral challenge.Mice were nasally immunised with 30 lg rTgUPase or PBS.Two weeks after the last immunisation, mice were orally challenged with tachyzoites.(A) Mice from two groups (n = 8 PBS and 8 rTgUPase) were orally infected with 1 Â 10 4 tachyzoites of the T. gondii RH strain.Liver and brain tachyzoite burdens were evaluated one month after the challenge.(B) Mouse survival rates of the two groups (n = 12 PBS and 12 rTgUPase) were monitored daily after challenge with 4 Â 10 4 tachyzoites of the T. gondii RH strain until Day 30 post-challenge.Differences in survival were significant (p < 0.01).These results are representative of two independent experiments.Values are the means ± SDs. ** p < 0.01.PBS, phosphate buffered saline.

Table 1 .
Lymphocyte proliferation and cytokine production by splenocytes stimulated with rTgUPase.